면역결핍 바이러스의 효소 integrase의 endonucleolytic activity

신차균 中央大學校 遺傳工學硏究所 1995 遺傳工學硏究論集 Vol.8 No.1

면역결핍바이러스의 integrase 단백질은 바이러스 DNA 유전자를 감염세포의 유전자에 공유결합으로 끼워넣어 바이러스 유전자의 복제와 바이러스의 생성을 가능하게하는 바이러스 증식에 필요불가결한 효소이다. 본연구에서는 박테리아에서 overexpression으로 생산한 integrase 단백질의 endonucleolytic 활성의 발현 조건을 조사하였다. 방사능으로 표식된 이중나선의 oligonucleotide 기질 0.1 pmol를 integrase 30 pmol로 처리하였을 때, 90분 정도의 반응시간에서 대부분의 기질이 선택적으로 절단되었다. 또한 이러한 절단반응에 조효소로써 Mg++이온과 Mn++이온이 필요한가를 각 이온의 여러 농도에서 조사해보았을 때, Mn++이온의 존재는 endonucleolytic 활성을 위하여는 꼭 필요하며, 최고 20 mM까지 효소활성이 나타나게하였다. 그러나, Mg++이온은 조사된 모든 농도에 있어서 효소활성이 관측되지 않았다. 또한 방사능으로 표식되지않은 경쟁 oligonucleotide를 사용하여 효소활성의 저해를 조사하였을 때, 1 ug이상의 oligonucleotide는 효과적으로 반응을 억제하였다. The integrase protein mediates insertion of viral DNA into cellular DNA that is essential for viral replication and virion production. Using the human immunodeficiency virus type-1(HIV-1) integrase protein prepared from the overexpressed bacteria, basic conditions for evaluation of the endonucleolytic activity were studied in vitro. Short duplex oligonucleotides corresponding to the ends of linear viral DNA were used as substrates. Endonucleolytic cleavages in a reaction containing 0.1 pmol oligonucleotide as substrate and 30 pmol integrase were almost completed in 90 min. Mn++ was absolutely required for the endonucleolytic activity of the HIV-1 integrase while Mg++ was not. The endonucleolytic activities were observed in the presence of 0.2 to 20 mM Mn++. However, the endonucleolytic activity was not detected in any concentrations of Mg++ in the absence of Mn++. Furthermore, the endonucleolytic activity was fairly inhibited in the presence of competitive oligonucleotides. T

면역결핍바이러스의 감염 진단법의 현황

심규부,신차균 中央大學校 遺傳工學硏究所 1996 遺傳工學硏究論集 Vol.9 No.1

국내에서는 아직 AIDS 환자의 수가 외국에 비해 적기 때문에 그만큼 AIDS대한 연구가 미국이나 유럽의 환자수가 많은 여러 선진국에 비하여 상당히 미약하다. 또한 진단시약의 개발도 부진해서 국내 몇 군데 안되는 곳에서 상품화 되어 시판되고 있으나 외국산 진단시약의 국내시장 점유울이 월등히 더 높다. 진단시약 개발 연구의 핵심은 특이도와 민감도를 둘 다 최대로 높혀서 가능한 위양성이나 위음성의 결과를 최소화 하는 데 있다. 그러나 아직까지 특이도와 민감도 둘 다 100%충족시킬 수 없다. 이런 현실 때문에 진단 기관의 특성에 따라서 더 비중을 두기도 하고, 특이도를 더 비중을 두기도한다. 적십자 기관과 같은 수혈을 담당하는 기관에서는 AIDS 환자의 피를 공급하면 안되기 때문에 당연히 민감도에 더 비중을 두게 된다. 현재 진단시약개발의 중점사상으로 감염초기 (window period)에 낮은 농도의 항체를 감지못하는 위음성(false negative)과 특히 Screening test에서 많은 위양성(false positive)의 문제점을 개선, 향상시켜서 좀 더 민감도(sensitivity)와 특이도(specificity)가 높은 진단용 시약이 개발되어야 할 것이다. 특히, 최근 보고에 의하면 외국산 진단 시약의 항체가 국내환자의 항원에 적합하지 않아서 잘 감지하지 못한다는 의견이 제기되고이있어 국내 AIDS 환자의 항원과 항체에 대한 연구를 바탕으로 국내 환자에 대한 적합한 진단시약 개발이 급선무이다. Current diagnosis for HIV infection is based on the observation that virus-specific antibodies are developed within few months in most persons infected with human immunodeficiency virus and thereafter during their lives. Many methods are used to detect HIV-specific antibodies in patient's sera or body fluids. These methods can be grouped into two types of test such Screening test and Confirm test. Elisa, agglutination, and dot blot methods are adapted for Screening test in which many samples are inspected relatively in short time. Indirect immunofluorescence assay, Western blot, and radioimmunoprecipitation assay used for Confirm test in which the samples reported as positive in Screening test are re-inspected, many false positive or negative in the tests due th the low specificity and sensitivity of the reagents, arising social and clinical problems.

Oct4 단백질의 DNA - 결합 영역에 융합된 HIV - 1 integrase

이민전,서진욱,김미라,신차균 中央大學校 遺傳工學硏究所 2002 遺傳工學硏究論集 Vol.15 No.1

Human immunodeficience virus type-1 (HIV-1) integrase (IN) mediates integration of viral DNA into the host cell genome which is an essential step during the life cycle of retroviruses. HIV-1 IN shows four enzymatic activities in vitro: non-specific DNA- binding, endonucleolytic (3'-processing), integration (strand transfer), and disintegration activities. Oct4 as a transcription factor recognizes and binds the specific sequences containing 5' -ATGCAAAT-3'. In order to study the specific binding and enzymatic activities of the integrase protein, we constructed the four different fusion proteins (OctIN1, OctIN2, INKOct1, INKOct1) using different combination of HIV-1 IN and Oct4 protein. The fusion proteins were overexpressed in Escherichia coli and purified by Ni-NTA and SP-sepharose columns. Analysis of the enzymatic activities showed that all the four fusion proteins has detectable endonucleolytic and integration activities, suggesting that the fusion proteins can be used to study derected integration and sequence specific DNA-binding of the integrase fusion protein.

스쿠알렌 합성 유전자를 도입한 형질전환 가시오가피

서진욱,정재훈,최용의,이학성,신차균 중앙대학교 유전공학연구소 2003 遺傳工學硏究論集 Vol.16 No.1

The enzyme, squalene synthase, represents a branch point in the isoprenoid pathway capable of diverting carbon flow specifically to the biosynthesis of phytosterol and Triterpenoid. Transgenic Eleutherococcus senticosus plants were prepared by introducing the genes for squalene synthase derived from Panax ginseng (PgSS1), hygromycin phosphotransferase (HPT) and green fluorescent protein (GFP) through Agrobacterium-mediated transformation. Establishment of transgenic plantlets were confirmed by the presence of PgSS1 and HPT bands in the genomic DNA preparation using a PCR method and a Southern blotting. In addition, expression of the introduced GFP DNA was confirmed by observing green fluorescence of GFP from the embroyos. In the in vitro analysis of the PgSS1 enzymatic activities the transgenic plants showed to have 1.5 to 3 times higher than wild type plant, indicating that the PgSS1 genes was well over-expressed in the transgenic plants. Especially the PgSS1 acivities of embroyogenic cell was 120 times higher than those of plantlet.

Symposium Abstracts ; Characteristics of HIV-1 Integrase as a Target of AntiAIDS Agent

(Cha Gyun Shin) 한국응용약물학회 1999 학술대회 Vol.1999 No.2

Beauvericin and enniatins H, I and MK1688 are new potent inhibitors of human immunodeficiency virus type-1 integrase

Shin, Cha-Gyun,An, Dog-Gn,Song, Hyuk-Hwan,Lee, Chan Springer Science and Business Media LLC 2009 The Journal of Antibiotics Vol.62 No.12

<P>Some enniatins (ENs) reportedly exhibit antiretroviral activities in vivo. The potential inhibitory activities of cyclic hexadepsipeptides such as beauvericin (BEA) and ENs H, I and MK1688 were investigated in vitro against human immunodeficiency virus type-1 (HIV-1) integrase and Moloney murine leukemia virus reverse transcriptase. BEA, EN I and EN MK1688 exhibited strong inhibitory activities against HIV-1 integrase, whereas EN H showed relatively weak activity. None of the examined compounds showed anti-reverse transcriptase activity. BEA was the most effective inhibitor of the tested cyclic hexadepsipeptides in inhibiting HIV-1 integrase. These results indicate the potential of cyclic hexadepsipeptides as a new class of potent inhibitors of HIV-1 integrase.The Journal of Antibiotics advance online publication, 6 November 2009; doi:10.1038/ja.2009.102.</P>

Biochemical Properties of Second Site Mutation of Human Immunodeficiency Virus Integrase

Shin,Cha-Gyun,Lee,Sang Kwang,Kim,Do-Jin,Oh,You-Take The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.6

A highly conserved amino acid, glutamic acid (Glu), present at position 152 in the catalytic domain of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein has been known to be critical for enzymatic function since substitution of Glu 152 with other residues results in a complete loss of enzymatic activities. In order to better understand the role of Glu 152 as a conserved residue in enzymatic action, intragenic second site mutations have been introduced around residue 152 of a mutant IN (E152A), and their biochemical properties were analyzed in terms of enzymatic activities. Disintegration activities were found to be significantly restored in several second site mutant INs, while integration activities were only recovered weakly. However, endonucleolytic activities were not discovered in all the mutant INs. These findings indicate that the second site mutations can partially restore that catalytic structure of the active site disturbed by the E152A mutation and lead to the regaining of integration and disintegration activities. In addition, it is also suggested that endonucleolytic activity requires a more accurate structure of the catalytic site than that for the integration and disintegration activities.

Biochemical Properties of Second Site Mutation of Human Immunodeficiency Virus Integrase

Shin, Cha Gyun,Kim, Do Jin,Oh, You Take,Lee, Sang Kwang 생화학분자생물학회 1998 BMB Reports Vol.32 No.6

Structural Changes in Human Immunodeficiency Virus Type 1 Mutant Integrase Proteins

Shin,Cha-Gyun 中央大學校 遺傳工學硏究所 1993 遺傳工學硏究論集 Vol.6 No.1

본 연구에서는 인간면역결핍 바이러스(HIV-1)의 유전자가 숙주세포의 유전자에 끼어들어 갈 때 작용하는 바이러스 효소(integrase)의 돌연변이 단백질의 구조변화에 대한 연구를 함으로써, 돌연변이 integrase를 갖는 바이러스의 복제와 형태가 비정상적인 것으로 판명된 앞선 연구결과에 대한 원인을 돌연변이 integrase 단백질구조 측면에서 이해하고자 하였다. 돌연변이 integrase 단백질을 in vitro transcription과 translation을 이용하여 생성하기 위하여, 돌연변이 proviral DNA로부터 PCR를 이용하여 돌연변이 integrase유전자를 expression vector에 cloning하였고, 이를 이용하여 돌연변이 integrase 단백질들을 생성하였다. 또한 이미 바이러스 integrase 단백질과 잘 결합하는 것으로 알려진 항체를 이용하여, immunoprecipitation하였을때, precipitation되는 정도가 비슷하여 돌연변이 integrase 단백질이 외형상으로는 wild type과 비슷한것으로 여겨지나, trypsin digestion 형태에서 S147I 돌연변이는 wild type과 달라서, 이 point mutation은 integrase단백질의 부분적인 구조를 바꾼 것으로 사료되며, 이러한 변화가 돌연변이바이러스의 복제불능을 유발할 수 있을 것으로 추측된다. In a previous work (Shin et al., 1993) several human immunodeficiency virus type 1 mutant viruses which have point mutation at integrase region showed defective viral replication, abortive infection or abnormal virion morphology. To understand these results in aspects of protein conformation, some of mutant integrase DNAs (wild type, D116A, S147I, and E152A) were cloned into expression vector by using PCR-amplification. Wild type and mutant integrase proteins were prepared through in vitro transcription and translation, respectively. All integrase proteins were immunoprecipitated by rabbit anti-integrase antiserum in same level, indicating that there is no dramatic changes in structure of all mutant proteins. However, partial digestion of the proteins with trypsin showed that cleavage pattern of S147I mutant protein was different from that of the wild type, suggesting that point mutation in the mutant protein may induce conformational change in local region and be further responsible for defective viral replication.

Biophysical Analysis of HIV-1 Virion and Core

Shin,Cha-Gyun 中央大學校 遺傳工學硏究所 1994 遺傳工學硏究論集 Vol.7 No.1

본 연구는 인간면역결핍 바이러스 (HIV-1)의 입자와 바이러스 핵의 물리화학적인 특성을 연구하기 위하여, 바이러스 입자를 비이온성 계면활성제로 처리하여 바이러스 입자구조에서 분리되는 바이러스 단백질들의 분포를 sucrose gradient를 사용하여 조사하였다. 바이러스 입자들을 비이온성 계면활성제인 NP40 0.01-10%로 처리할 대 바이러스 막에 존재하는 표면단백질 (surface protein)과 바이러스 핵을 형성하는 capsid 단백질은 거의 완전히 제거되었다. 그러나, 바이러스 막과 핵 사이에 존재하는 것으로 알려진 matrix 단백질은 바이러스 nucleocapsid 단백질과 함께 subviral structure로 남아있어, 바이러스 입자구조상에서 matrix 단백질이 nucleocapsid 단백질 또는 바이러스 유전자와 결합하고 있을 가능성을 제시하고있다. 또한 바이러스 입자를 2가 양이온 (Mg++) 존재하에 계면활성제로 처리하면, 상대적으로 높은 농도(5 mM)의 Mg++존재에서는 바이러스 입자가 완전히 분해되어, 인간면역결핍 바이러스의 입자와 핵을 구성하는 단백질들이 다른 retrovirus에서와는 달리 강하게 결합하고 있지 않는 것으로 추측된다. The fine structural changes of the HIV-1 virions caused by detergent were studied to understand structure of the HIV-1 core and virion in biophysical aspect. Threatment of the HIV-1 virion with non-ionic detergent NP40 of 0.01 to 10% caused complete loss of capsid protein (p24) and the membrane glycoprotein (gp120) from the viron structure, but matrix protein (p17) and nucleocapsid proteins remained with reverse transcriptase (RT, p66 and p51), integrase in the subviral structure designated as detergent core. In addition, HIV-1 viron was observed to be completely dissociated when it was treated with NP40 of 1.6% in the presence of 5mM MgCl₂, while more distinct detergent core was formed under same treatment using 0.1mM MgCl₂. Therefore, it is likely that weak association among viral proteins constructing core is responsible for easy inactivation of HIV-1 virion in vitro.